Escherichia coli surface display for the selection of nanobodies

Abstract

SummaryNanobodies (Nbs) are the smallest functional antibody fragments known in nature and have multiple applications in biomedicine or environmental monitoring. Nbs are derived from the variable segment of camelid heavy chain‐only antibodies, known as VHH. For selection, libraries of VHH gene segments from naïve, immunized animals or of synthetic origin have been traditionally cloned in E. coli phage display or yeast display systems, and clones binding the target antigen recovered, usually from plastic surfaces with the immobilized antigen (phage display) or using fluorescence‐activated cell sorting (FACS; yeast display). This review briefly describes these conventional approaches and focuses on the distinct properties of an E. coli display system developed in our laboratory, which combines the benefits of both phage display and yeast display systems. We demonstrate that E. coli display using an N‐terminal domain of intimin is an effective platform for the surface display of VHH libraries enabling selection of high‐affinity Nbs by magnetic cell sorting and direct selection on live mammalian cells displaying the target antigen on their surface. Flow cytometry analysis of E. coli bacteria displaying the Nbs on their surface allows monitoring of the selection process, facilitates screening, characterization of antigen‐binding clones, specificity, ligand competition and estimation of the equilibrium dissociation constant (KD).

Recombinant expression of nanobodies and nanobody-derived immunoreagents

“…Nanobodies fold into their native and functional structure once secreted at the surface of bacterial or yeast cells and nanobody display have been used for both screening purposes and for preparing wholecell biosensors [88][89][90][91]. The advantages represented by displaying functional macromolecules on cell surface are multiple and have been…”

Synthetic consortia of nanobody‐coupled and formatted bacteria for prophylaxis and therapy interventions targeting microbiome dysbiosis‐associated diseases and co‐morbidities

“…14 kDa) and superior biophysical and antigen-binding properties of Nbs make them ideal candidates for diverse applications requiring the selective recognition and binding to Ags of different entities, including viruses, bacteria, and eukaryotic cell targets (Oliveira et al, 2013;Chakravarty et al, 2014;Steeland et al, 2016;Van Audenhove and Gettemans, 2016). Importantly, high affinity Nbs of desired specificity can be selected from libraries of VHH gene segments, amplified from peripheral blood lymphocytes of immunized animals, and cloned and expressed in bacteriophages, E. coli or yeast cells (Salema and Fernandez, 2017).…”

Ultrahigh‐throughput screening system for directed polymer binding peptide evolution

“…Both, antibodies as wells as scaffold proteins have been engineered applying yeast display technologies as summarized in two comprehensive reviews by Boder, Raeeszadeh‐Sarmazdeh and Price () and Konning and Kolmar (). Yeast display systems commonly contain library sizes of up to 10 9 mutants, but are comparably time‐consuming due to the growth rate of yeast cells (Benatuil, Belk, & Hsieh, ; Salema & Fernandez, ). Bacterial display systems are routinely used for the improvement of enzymes, antibodies, nanobodies, or peptides (Bidlingmaier & Liu, ; Jose & Zangen, ; Salema & Fernandez, ).…”

“…Yeast display systems commonly contain library sizes of up to 10 9 mutants, but are comparably time‐consuming due to the growth rate of yeast cells (Benatuil, Belk, & Hsieh, ; Salema & Fernandez, ). Bacterial display systems are routinely used for the improvement of enzymes, antibodies, nanobodies, or peptides (Bidlingmaier & Liu, ; Jose & Zangen, ; Salema & Fernandez, ). One frequently used bacterial cell surface system is based on the esterase EstA of Pseudomonas aeruginosa which has successfully been applied to present functional enzymes or hybrid catalysts on the E. coli surface (e.g., LipA [ Bacillus subtilis ] or cutinase [ Fusarium solani pisi ]; S. Becker, Heppeler, Wentzel, Jaeger, & Kolmar, ) or metathesis catalysts (Grimm, Polen, Hayashi, & Schwaneberg, ).…”